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The Hasmonaean rulers struck coins, exclusively in bronze, beginning with John Hyrcanus I (135–104 BCE; Hendin 2020). Their most common denomination was a coin with a diameter averaging 15 mm and c...

Aims/hypothesis Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. Methods CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. Results Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. Conclusions/interpretation Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. Data availability The datasets generated and/or analysed during the current study are available in the Biorxiv repository (www.biorxiv.org/content/10.1101/2020.05.18.099200v1). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576) and ProteomeXchange (www.ebi.ac.uk/pride/archive/projects/PXD021597) repositories, respectively. Graphical abstract

AIM: To determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN) channels impact glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) modulation of islet Ca(2+) handling and insulin secretion. METHODS: The impact of liraglutide (GLP-1 analogue) on islet Ca(2+) handling, HCN currents and insulin secretion was monitored with fluorescence microscopy, electrophysiology and enzyme immunoassays, respectively. Furthermore, liraglutide-mediated β-to-δ-cell cross-communication was assessed following selective ablation of either mouse islet δ or β cells. RESULTS: Liraglutide increased β-cell Ca(2+) oscillation frequency in mouse and human islets under stimulatory glucose conditions. This was dependent in part on liraglutide activation of HCN channels, which also enhanced insulin secretion. Similarly, liraglutide activation of HCN channels also increased β-cell Ca(2+) oscillation frequency in islets from rodents exposed to a diabetogenic diet. Interestingly, liraglutide accelerated Ca(2+) oscillations in a majority of islet δ cells, which showed synchronized Ca(2+) oscillations equivalent to β cells; therefore, we assessed if either cell type was driving this liraglutide-mediated islet Ca(2+) response. Although δ-cell loss did not impact liraglutide-mediated increase in β-cell Ca(2+) oscillation frequency, β-cell ablation attenuated liraglutide-facilitated acceleration of δ-cell Ca(2+) oscillations. CONCLUSION: The data presented here show that liraglutide-induced stimulation of islet HCN channels augments Ca(2+) oscillation frequency. As insulin secretion oscillates with β-cell Ca(2+), these findings have important implications for pulsatile insulin secretion that is probably enhanced by liraglutide activation of HCN channels and therapeutics that target GLP-1Rs for treating diabetes. Furthermore, these studies suggest that liraglutide as well as GLP-1-based therapies enhance δ-cell Ca(2+) oscillation frequency and somatostatin secretion kinetics in a β-cell-dependent manner.

Despite the advantages of CO2 electrolyzers, efficiency losses due to mass and ionic transport across the membrane electrode assembly (MEA) are critical bottlenecks for commercial-scale implementation. In this study, more efficient electrolysis of CO2 was achieved by increasing cation exchange membrane (CEM) hydration via the humidification of the CO2 reactant inlet stream. A high current density of 755 mA/cm2 was reached by humidifying the reactant CO2 in a MEA electrolyzer cell featuring a CEM. The power density was reduced by up to 30% when the fully humidified reactant CO2 was introduced while operating at a current density of 575 mA/cm2. We reduced the ohmic losses of the electrolyzer by fourfold at 575 mA/cm2 by fully humidifying the reactant CO2. A semiempirical CEM water uptake model was developed and used to attribute the improved performance to 11% increases in membrane water uptake and ionic conductivity. Our CEM water uptake model showed that the increase in ohmic losses and the limitation of ionic transport were the result of significant dehydration at the central region of the CEM and the anode gas diffusion electrode-CEM interface region, which exhibited a 2.5% drop in water uptake.

Proton exchange membrane fuel cells (PEMFCs), fed with oxygen from ambient air and hydrogen, spontaneously produce power. However, key materials, gas diffusion layers, catalysts, ionomers, and membranes, are sensitive to a multitude of other species that may be inadvertently introduced into PEMFCs even at parts per million (ppm) or less levels. For a commercially relevant catalyst loading of 0.1 mg Pt cm–2 and a severe case such as 100 ppm acetylene in air, the temporary cell voltage loss exceeds 80 % [1]. Exposure to species such as bromomethane is even more problematic because the cell voltage cannot be restored without a specific recovery procedure [2]. The development of preventive and recovery procedures to improve durability is facilitated by contamination mechanisms. Although mass transfer losses were often observed in the presence of organic species [1], the commonest contaminants either in air or released by system components [3], the origin of this effect is currently unclear. It may stem from adsorption on carbon [4] or platinum [5] respectively modifying surface hydrophobicity and liquid water management or reducing the catalyst active surface, which increases the real current density. Several diagnostic techniques able to either characterize water management or oxygen mass transfer were used to evaluate PEMFCs contaminated with propene or methyl methacrylate. Neutron radiographs were obtained for both in-plane and through-plane cell configurations to record changes in liquid water distribution [6,7]. Polarization curves were measured with O2, 21 % O2 in He, and air to derive concentration, molecular diffusion and ionomer permeability mass transfer overpotentials [8]. Overall oxygen mass transfer coefficients were acquired by first operating PEMFCs fed with 1 % to 7 % O2 in He, N2 or CO2 streams under limiting current conditions. Subsequently, a mathematical model was used to fit average limiting current data as a function of oxidant flow rate to extract the overall oxygen mass transfer coefficient. Separation of the overall mass transfer coefficient into molecular and Knudsen diffusion, and ionomer permeability contributions was conducted by plotting mass transfer resistance data (inverse of the mass transfer coefficient) either as a function of the O2 diluent molecular weight or O2 concentration and extrapolating linear correlations to the origin. The original separation method [9] was improved by taking advantage of the insensitivity of Knudsen diffusion to variations in O2 concentration. Propene and methyl methacrylate were selected because they have largely different solubility in water (surrogate hydrophobicity measure), minimize test durations and simplify test procedures (fast contamination and recovery kinetics), and facilitate data interpretation (minimal current distribution and effective concentration changes). Experimental data demonstrated that changes in liquid water distributions were subtle and cannot explain mass transfer losses induced by contaminants. In contrast, ionomer permeability overpotentials and mass transfer coefficients revealed major increases in the presence of both contaminants. These observations support the notion that propene and methyl methacrylate contaminants adsorb on the Pt catalyst, decrease the active area, and bring the real current density closer to the limiting value. Experimental data analyses based on a contamination model that include catalyst surface processes for both O2 and contaminant (adsorption, reaction, desorption) [10] confirmed this hypothesis. The proposed contamination mechanism is similar to the increase in oxygen mass transfer overpotential induced by a decrease in Pt catalyst loading [11]. [1] J. St-Pierre, Y. Zhai, Molecules, 25 (2020) article 1060. [2] Y. Zhai, O. Baturina, D. Ramaker, E. Farquhar, J. St-Pierre, K. Swider-Lyons, Electrochim. Acta, 213 (2016) 482. [3] J. St-Pierre, M. Angelo, K. Bethune, J. Ge, S. Higgins, T. Reshetenko, M. Virji, Y. Zhai, Electrochem. Soc. Trans., 61(23) (2014) 1. [4] X. Zhang, B. Gao, A. E. Creamer, C. Cao, Y. Li, J. Hazard. Mater., 338 (2017) 102. [5] Y. Itagaki, M. Mori, Y. Sadaoka, Curr. Opin. Electrochem., 11 (2018) 72. [6] D. S. Hussey, D. L. Jacobson, M. Arif, K. J. Coakley, D. F. Vecchia, J. Fuel Cell Sci. Technol., 7 (2010) article 021024. [7] D. S. Hussey, D. Spernjak, A. Z. Weber, R. Mukundan, J. Fairweather, E. L. Brosha, J. Davey, J. S. Spendelow, D. L. Jacobson, R. L. Borup, J. Appl. Phys., 112 (2012) article 104906. [8] J. St-Pierre, M. Angelo, K. Bethune, J. Huizingh, T. Reshetenko, M. Virji, Y. Zhai, Modern Fuel Cell Testing Laboratory, in Springer Handbook of Electrochemical Energy, Part D, Chapter 19, Edited by C. Breitkopf, K. Swider-Lyons, Springer, 2017, p. 611. [9] T. Reshetenko, J. St-Pierre, J. Electrochem. Soc., 161 (2014) F1089. [10] J. St-Pierre, J. Electrochem. Soc., 156 (2009) B291. [11] A. Z. Weber, A. Kusoglu, J. Mater. Chem. A, 2 (2014) 17207.

Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are coproduced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine cell function. Mice with loss of Sin3a in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca2+ influx of Sin3a-deficient β-cells. RNA sequencing coupled with candidate chromatin immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in β-cells, which modulate Ca2+/ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Finally, mice with loss of both Sin3a and Sin3b in multipotent embryonic pancreatic progenitors had significantly reduced islet cell mass at birth, caused by decreased endocrine progenitor production and increased β-cell death. These findings highlight the stage-specific requirements for the presumed “general” coregulators Sin3a and Sin3b in islet β-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival.

The PEF was founded as a charity, relying almost entirely on public generosity for its funding. Its insecure financial basis was at odds with its ambitious programme of surveys and excavations, whi...

The activation of neuronal plasma membrane Ca2+channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca2+channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues949Arg-Arg-Lys951to three alanines disrupts CaMKII bindingin vitroand CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENTPrecise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.

Lithium-ion batteries have received significant research interest due to their advantages in energy and power density, which are important to enabling many devices. One route to further increase energy density is to fabricate thicker electrodes in the battery cell; however, careful consideration must be taken when designing electrodes as to how increasing the thickness impacts the multiscale and multiphase molecular transport processes, which can limit the overall battery operating power. Design of these electrodes necessitates probing the molecular processes when the battery cell undergoes electrochemical charge/discharge. One tool for in situ insights into the cell is neutron imaging, because neutron imaging can provide information of where electrochemical processes occur within the electrodes. In this manuscript, neutron imaging is applied to track the lithiation/delithiation processes within electrodes at different current densities for a full cell with a thick sintered Li(4)Ti(5)O(12) anode and LiCoO(2) cathode. The neutron imaging reveals that the molecular distribution of Li(+) during discharge within the electrode is sensitive to the current density, or equivalently discharge rate. An electrochemical model provides additional insights into the limiting processes occurring within the electrodes. In particular, the impact of tortuosity and molecular transport in the liquid phase within the interstitial regions in the electrodes are considered, and the influence of tortuosity was shown to be highly sensitive to the current density. Qualitatively, the experimental results suggest that the electrodes behave consistent with the packed hard sphere approximation of Bruggeman tortuosity scaling, which indicates that the electrodes are largely mechanically intact but also that a design that incorporates tunable tortuosity could improve the performance of these types of electrodes.

We survey several neutron imaging and diffraction methods for non-destructive testing and evaluation of intact, commercial lithium-ion batteries. Specifically, far-field interferometry was explored as an option to probe a wide range of autocorrelation lengths within the batteries via neutron imaging. The dark-field interferometry images change remarkably from fresh to worn batteries, and from charged to discharged batteries. When attempting to search for visual evidence of battery degradation, neutron Talbot-Lau grating interferometry exposed battery layering and particle scattering through dark-field imaging. Bragg edge imaging also reveals battery wear and state of charge. Neutron diffraction observed chemical changes between fresh and worn, charged and discharged batteries. However, the utility of these methods, for commercial batteries, is dependent upon battery size and shape, with 19 to 43 mAh prismatic batteries proving most convenient for these experimental methods. This study reports some of the first spatially resolved, small angle scattering (dark-field) images showing battery degradation.

Water retention curves are essential for understanding the hydrologic behavior of partially saturated porous media and modeling flow and transport processes within the vadose zone. We directly measured the main drying and wetting branches of the average water retention function obtained using two-dimensional neutron radiography. Flint sand columns were saturated with water and then drained and rewetted under quasi-equilibrium conditions using a hanging water column setup. Digital images (2048 by 2048 pixels) of the transmitted flux of neutrons were acquired at each imposed matric potential (∼10–15 matric potential values per experiment) at the National Institute of Standards and Technology Center for Neutron Research BT-2 neutron imaging beam line. Volumetric water contents were calculated on a pixel-by-pixel basis using Beer-Lambert’s law after taking into account beam hardening and geometric corrections. To account for silica attenuation and remove scattering effects at high water contents, the volumetric water contents were normalized (to give relative saturations) by dividing the drying and wetting sequences of images by the images obtained at saturation and satiation, respectively. The resulting pixel values were then averaged and combined with information on the imposed basal matric potentials to give average water retention curves. The average relative saturations obtained by neutron radiography showed an approximate one-to-one relationship with the average values measured volumetrically using the hanging water column setup. There were no significant differences (P < 0.05) between the parameters of the van Genuchten equation fitted to the average neutron radiography data and those estimated from replicated hanging water column data. Our results indicate that neutron imaging is a very effective tool for quantifying the average water retention curve.

We report on efforts to improve the achievable spatial resolution in neutron imaging by centroiding the scintillation light from gadolinium oxysulfide scintillators. The current state-of-the-art neutron imaging spatial resolution is about 10 μm, and many applications of neutron imaging would benefit from at least an order of magnitude improvement in the spatial resolution. The detector scheme that we have developed magnifies the scintillation light from a gadolinium oxysulfide scintillator, calculates the center of mass of the scintillation event, resulting in an event-based imaging detector with spatial resolution of about 2 μm.

Platinum group metal-free (PGM-free) catalysts are a low-cost alternative to expensive PGM catalysts for polymer electrolyte fuel cells. However, due to the low volumetric activity of PGM-free catalysts, the catalyst layer thickness of the PGM-free catalyst electrode is an order of magnitude higher than PGM based electrodes. The thick PGM-free electrodes suffer from increased transport resistance and poor water management, which ultimately limits the fuel cell performance. This manuscript presents the study of water management in the PGM-free electrodes to understand the transport limitations and improve fuel cell performance. In-operando neutron imaging is performed to estimate the water content in different components across the fuel cell thickness. Water saturation in thick PGM electrodes, with similar catalyst layer thickness to PGM-free electrodes, is lower than in the PGM-free electrodes irrespective of the operating conditions, due to high water retention by PGM-free catalysts. Improvements in fuel cell performance are accomplished by enhancing water removal from the flooded PGM-free electrode in three ways: (i) enhanced water removal with a novel microporous layer with hydrophilic pathways incorporated through hydrophilic additives, (ii) water removal through anode via novel GDL in the anode, and (iii) lower water saturation in PGM-free electrode structures with increased catalyst porosity.

Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on β-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in βTC3 cells, a murine pancreatic β-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to ∼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in β-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.

Models of fluid flow are used to improve the efficiency of oil and gas extraction and to estimate the storage and leakage of carbon dioxide in geologic reservoirs. Therefore, a quantitative understanding of key parameters of rock–fluid interactions, such as contact angles, wetting, and the rate of spontaneous imbibition, is necessary if these models are to predict reservoir behavior accurately. In this study, aqueous fluid imbibition rates were measured in fractures in samples of the Eagle Ford Shale using neutron imaging. Several liquids, including pure water and aqueous solutions containing sodium bicarbonate and sodium chloride, were used to determine the impact of solution chemistry on uptake rates. Uptake rate analysis provided dynamic contact angles for the Eagle Ford Shale that ranged from 51 to 90° using the Schwiebert–Leong equation, suggesting moderately hydrophilic mineralogy. When corrected for hydrostatic pressure, the average contact angle was calculated as 76 ± 7°, with higher values at the fracture inlet. Differences in imbibition arising from differing fracture widths, physical liquid properties, and wetting front height were investigated. For example, bicarbonate-contacted samples had average contact angles that varied between 62 ± 10° and ∼84 ± 6° as the fluid rose in the column, likely reflecting a convergence–divergence structure within the fracture. Secondary imbibitions into the same samples showed a much more rapid uptake for water and sodium chloride solutions that suggested alteration of the clay in contact with the solution producing a water-wet environment. The same effect was not observed for sodium bicarbonate, which suggested that the bicarbonate ion prevented shale hydration. This study demonstrates how the imbibition rate measured by neutron imaging can be used to determine contact angles for solutions in contact with shale or other materials and that wetting properties can vary on a relatively fine scale during imbibition, requiring detailed descriptions of wetting for accurate reservoir modeling.

Transitional hypoglycemia in normal newborns occurs in the first 3 days of life and has clinical features consistent with hyperinsulinism. We found a lower threshold for glucose-stimulated insulin secretion from freshly isolated embryonic day (E) 22 rat islets, which persisted into the first postnatal days. The threshold reached the adult level by postnatal day (P) 14. Culturing P14 islets also decreased the glucose threshold. Freshly isolated P1 rat islets had a lower threshold for insulin secretion in response to 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid, a nonmetabolizable leucine analog, and diminished insulin release in response to tolbutamide, an inhibitor of β-cell KATP channels. These findings suggested that decreased KATP channel function could be responsible for the lower glucose threshold for insulin secretion. Single-cell transcriptomic analysis did not reveal a lower expression of KATP subunit genes in E22 compared with P14 β cells. The investigation of electrophysiological characteristics of dispersed β cells showed that early neonatal and cultured cells had fewer functional KATP channels per unit membrane area. Our findings suggest that decreased surface density of KATP channels may contribute to the observed differences in glucose threshold for insulin release.

Maturity-onset diabetes of the young (MODY) is a heterogeneous group of monogenic disorders of impaired pancreatic β cell function. The mechanisms underlying MODY include β cell KATP channel dysfunction (e.g., KCNJ11 [MODY13] or ABCC8 [MODY12] mutations); however, no other β cell channelopathies have been associated with MODY to date. Here, we have identified a nonsynonymous coding variant in KCNK16 (NM_001135105: c.341T>C, p.Leu114Pro) segregating with MODY. KCNK16 is the most abundant and β cell–restricted K+ channel transcript, encoding the two-pore-domain K+ channel TALK-1. Whole-cell K+ currents demonstrated a large gain of function with TALK-1 Leu114Pro compared with TALK-1 WT, due to greater single-channel activity. Glucose-stimulated membrane potential depolarization and Ca2+ influx were inhibited in mouse islets expressing TALK-1 Leu114Pro with less endoplasmic reticulum Ca2+ storage. TALK-1 Leu114Pro significantly blunted glucose-stimulated insulin secretion compared with TALK-1 WT in mouse and human islets. These data suggest that KCNK16 is a previously unreported gene for MODY.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control human β-cell insulin secretion. Here, we examined if HCN channels impact glucagon-like-peptide 1 (GLP-1) receptor modulation of islet Ca2+ handling. We found that Liraglutide (100nM LIRA), a GLP-1 analog, increased mouse β-cell Ca2+ oscillation frequency and cAMP levels in high (11 mM) but not low glucose. Similarly, a subset of human β-cells (65%) show LIRA-induced elevations in Ca2+ oscillation frequency (by 70%, P Disclosure K. E. Zaborska: None. K. L. Jordan: None. P. Dadi: None. M. Dickerson: None. S. M. Graff: None. D. Jacobson: None.

Perturbations in Ca2+ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K+ countercurrent for [Ca2+]ER release, thereby tuning [Ca2+]ER storage and release. During β-cell Ca2+ influx, TALK-1-enhanced [Ca2+]ER release leads to activation of the hyperpolarizing Ca2+ activated K+ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca2+ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca2+ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI+) flux assay (z’ =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca2+]ER release (e.g. K2P16-i7(IC50=5.3μM); 63.36% [Ca2+]ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca2+]ER storage as well as cytosolic Ca2+ oscillations. TALK-1 inhibitors significantly increased islet [Ca2+]ER storage and Ca2+ oscillation frequency (e.g.K2P16-i7; [Ca2+]ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca2+]ER AUC=49.7a.u., peaks/min= 0.17, p-value Disclosure A. Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None.

Abstract Fuel cells have potential for small power applications that require long periods, as much as several decades, of uninterrupted power. Small microwatt fuel cell stacks of varying membrane thickness were designed to allow for passive water management. The passive water management system was incorporated into the stack design by use of exposed Nafion membrane for lateral water transport. 1 An experimental and theoretical study of water management across the operating temperature range was conduction, including operando neutron imaging and the development of a multiphysics two-dimensional model. Stack testing was done between -55 °C to 85 °C using a baseline output current of 10 µA and periodic pulses of 4.5 mA lasting 100 ms, every 100 s, or an average current of 16 µA. The stack spatial water concentration was monitored during various operating conditions through in situ neutron imaging. Figure 1 shows the main points of interest on the stack. Operating temperatures ranged from -55 °C to 85 °C to analyze water formation and subsequent water removal. Freezing and below-freezing temperatures were controlled by use of a liquid nitrogen cooled series of insulated boxes to prevent external water condensation during imaging. These measurements suggest successful water transport laterally, enabling operation from the sub-freezing environment to temperatures near the boiling point of water. Figure 2a depicts a constant operation for two hours at 16 µA and -20 ºC and the subsequent drying process in Figure 2b. Water production due to gas crossover in the stacks was quantified at below-freezing temperatures. At temperatures at and below -20 °C, the water production due to gas crossover was greater than the amount of water removal via the passive water management strategy with the current turned off, rather than a drying of the cell as expected. With the gases turned off as well, drying occurs. This indicates that the cell design must allow for water (ice) build-up during operation at these sub-freezing temperatures for successful operation. To avoid large amounts of hydrogen loss due to membrane crossover, we employ multiple layers of membrane to make a thick, mechanically reinforced and chemically stabilized membrane. A two-dimensional multiphysics model for evaluation of water management was also developed to further describe and explore the passive water management system under different conditions. The fundamental model in its present form shows a similar shape but indicates faster water build-up than experimental data. This discrepancy is currently being resolved to allow for a more accurate representation. Acknowledgments: This work was supported by the U.S. Department of Energy. References: Chintam, M. S. Wilson, T. Rockward, S. Stariha, A. M. Baker, E. L. Brosha, D. S. Hussey, J. M. LaManna, D. L. Jacobson, J. Rau, R. L. Borup, ECS Transactions 86 (13), 233-244. Figure 1

Impaired pancreatic {beta}-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of endocrine cells. MiR-125b is a highly conserved miRNA abundant in {beta}-cells, though its role in these cells remains unclear. Here, we show that miR-125b expression in human islets correlates with body mass index (BMI) of the donors and is regulated by glucose in an AMP-activated protein kinase-dependent manner in both mice and humans. Using and unbiased high-throughput approach, we identify dozens of direct gene targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Whereas inactivation of miR-125b in human beta cells led to shorter mitochondria and improved glucose stimulated insulin secretion, mice over-expressing mir-125b selectively in {beta}-cells displayed defective insulin secretion and marked glucose intolerance. Moreover, the {beta}-cells of these transgenic animals showed strongly reduced insulin content and secretion and contained enlarged lysosomal structures. Thus, miR125b provides a glucose-controlled regulator of organelle dynamics that negatively regulates insulin secretion in {beta}-cells. HighlightsO_LIIslet miR125b correlates with BMI and is regulated by glucose via AMP-activated protein kinase in {beta}-cells C_LIO_LImiR125b targets dozens of genes including those involved in the regulation of mitochondrial (i.e. Mtfp1) and lysosomal (i.e. M6pr) morphology/function C_LIO_LIDeletion of miR125b results in shorten mitochondria and improves glucose stimulated insulin secretion (GSIS) C_LIO_LISelective overexpression of miR125b in {beta}-cells causes the appearance of enlarged lysosomes, reduced GSIS and insulin content, loss of {beta}-cell identity and impaired insulin secretion and glucose tolerance in vivo C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/444559v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1772ccdorg.highwire.dtl.DTLVardef@11e6e76org.highwire.dtl.DTLVardef@e62b81org.highwire.dtl.DTLVardef@132405c_HPS_FORMAT_FIGEXP M_FIG C_FIG

A heterozygous missense mutation producing a variant of the islet β-cell- enriched MAFA transcription factor (p.Ser64Phe (S64F)) was identified in patients who developed adult-onset, β-cell dysfunction (diabetes or insulinomatosis), with men more prone to diabetes than women. This mutation engenders increased stability to the normally unstable MAFA protein. To obtain insight into how this variant impacts β cell function, we developed a mouse model expressing S64F MafA and found sex- dependent phenotypes, with heterozygous mutant males (MafAS64F/+) displaying impaired glucose tolerance while females were slightly hypoglycemic with improved blood glucose clearance. Only MafAS64F/+ males showed transiently higher MafA protein levels preceding the onset of glucose intolerance and sex-dependent, differential expression of genes involved in Ca2+ signaling, DNA damage, aging, and senescence. Functional changes in islet Ca2+ handling and signs of islet aging and senescence processes were uniquely observed in male animals. In addition, MAFAS64F expression in male human β cells accelerated cellular senescence and increased production of WT senescence-associated secretory proteins compared to cells expressing MAFA Together, these results implicate a conserved mechanism of accelerated islet aging and senescence in promoting diabetes in MAFAS64F carriers in a sex-dependent manner.

Objective Elevations in pancreatic α-cell intracellular Ca2+ ([Ca2+]i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca2+ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K+ (KATP) channels in cardiomyocytes, lactate may also modulate α-cell KATP. Therefore, this study investigated how lactate signaling controls α-cell Ca2+ handling and GCG secretion. Methods Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca2+ handling, Vm, KATP currents, and GCG secretion. Results Lactate-inhibited mouse (75 ± 25%) and human (47 ± 9%) α-cell [Ca2+]i fluctuations only under low-glucose conditions (1 mM) but had no effect on β- or δ-cells [Ca2+]i. Glyburide inhibition of KATP channels restored α-cell [Ca2+]i fluctuations in the presence of lactate. Lactate transport into α-cells via MCTs hyperpolarized mouse (14 ± 1 mV) and human (12 ± 1 mV) α-cell Vm and activated KATP channels. Interestingly, pyruvate showed a similar KATP activation profile and α-cell [Ca2+]i inhibition as lactate. Lactate-induced inhibition of α-cell [Ca2+]i influx resulted in reduced GCG secretion in mouse (62 ± 6%) and human (43 ± 13%) islets. Conclusions These data demonstrate for the first time that lactate entry into α-cells through MCTs results in KATP activation, Vm hyperpolarization, reduced [Ca2+]i, and inhibition of GCG secretion. Thus, taken together, these data indicate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function., Graphical abstract Image 1, Highlights • Lactate reduces islet α-cell Ca2+ entry under low glucose conditions. • Lactate does not alter β- or δ-cell Ca2+ handling under low glucose conditions. • Lactate enters islet α-cells through monocarboxylate transporters. • Lactate hyperpolarizes islet α-cell membrane potential by activating KATP channels. • Lactate reduces mouse and human islet glucagon secretion.

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Neutrons and X-rays provide excellent complementary, nondestructive probes to understand internal structure of systems across engineering and material science. With its sensitivity to hydrogen, neutrons excel at separating fluids, such as water or oil, from solid and gas phases in three-phase systems whereas X-rays excel at identifying the solid phase. To fully leverage the complementarity of the two methods, the National Institute of Standards and Technology (NIST) has developed the Neutron and X-ray Tomography (NeXT) system which orients a microfocus X-ray generator orthogonally to a reactor sourced thermal neutron beam. This orientation allows for truly simultaneous acquisition of both modalities so that the multi-modal data sets of samples that are evolving with time or undergoing stochastic processes can be directly correlated. The NeXT system has been available for external researchers since 2015 through the NIST Center for Neutron Research user facility program. Significant efforts have resulted in distributable software packages to facilitate image denoising, tomography reconstruction, volume registration, and bivariate histogram segmentation. This paper will give an overview of the NeXT system, explain the process for bivariate histogram segmentation, and provide several examples of use cases for the system.

Key points Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca2+ ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how β-cell TSPAN-7 regulates Ca2+ handling and hormone secretion. We find that TSPAN-7 reduces β-cell glucose-stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls β-cell function through a direct interaction with L-type voltage-dependent Ca2+ channels (CaV 1.2 and CaV 1.3), which reduces channel Ca2+ conductance. TSPAN-7 slows activation of CaV 1.2 and accelerates recovery from voltage-dependent inactivation; TSPAN-7 also slows CaV 1.3 inactivation kinetics. These findings strongly implicate TSPAN-7 as a key regulator in determining the set-point of glucose-stimulated Ca2+ influx and insulin secretion. Abstract Glucose-stimulated insulin secretion (GSIS) is regulated by calcium (Ca2+ ) entry into pancreatic β-cells through voltage-dependent Ca2+ (CaV ) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling, and thus they may also modulate GSIS. TSPAN-7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN-7 is highly expressed in β- and α-cells; however, the function of islet TSPAN-7 has not been determined. Here, we show that TSPAN-7 knockdown (KD) increases glucose-stimulated Ca2+ influx into mouse and human β-cells. Additionally, mouse β-cell Ca2+ oscillation frequency was accelerated by TSPAN-7 KD. Because TSPAN-7 KD also enhanced Ca2+ entry when membrane potential was clamped with depolarization, the effect of TSPAN-7 on CaV channel activity was examined. TSPAN-7 KD enhanced L-type CaV currents in mouse and human β-cells. Conversely, heterologous expression of TSPAN-7 with CaV 1.2 and CaV 1.3 L-type CaV channels decreased CaV currents and reduced Ca2+ influx through both channels. This was presumably the result of a direct interaction of TSPAN-7 and L-type CaV channels because TSPAN-7 coimmunoprecipitated with both CaV 1.2 and CaV 1.3 from primary human β-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human β-cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN-7 modulation of β-cell L-type CaV channels is a key determinant of β-cell glucose-stimulated Ca2+ entry and thus the set-point of GSIS.

The melastatin subfamily of the transient receptor potential channels (TRPM) are regulators of pancreatic β-cell function. TRPM7 is the most abundant islet TRPM channel; however, the role of TRPM7 in β-cell function has not been determined. Here, we utilized various spatiotemporal transgenic mouse models to investigate how TRPM7 knockout influences pancreatic endocrine development, proliferation, and function. Ablation of TRPM7 within pancreatic progenitors reduced pancreatic size, as well as α-cell and β-cell mass. This resulted in impaired glucose tolerance due to decreased serum insulin levels. However, ablation of TRPM7 following endocrine specification or in adult mice did not impact endocrine expansion or glucose tolerance. As TRPM7 regulates cell proliferation, we assessed how TRPM7 influences β-cell hyperplasia under insulin resistant conditions. β-cell proliferation induced by high-fat diet was significantly decreased in TRPM7 deficient β-cells. The endocrine roles of TRPM7 may be influenced by cation flux through the channel, and indeed we find that TRPM7 ablation alters β-cell intracellular Mg2+. Together, these findings reveal that TRPM7 controls pancreatic progenitor expansion and β-cell proliferation, which is likely due to regulation of Mg2+homeostasis.SummaryThis manuscript identifies a critical developmental role for TRPM7 channels in pancreatic progenitor cells. The manuscript also determines that TRPM7 plays a key role in β-cell proliferation under insulin-resistant conditions.